Telomeres protect the natural ends of linear chromosomes from recognition by the DNA damage response machinery. Not surprisingly, critically short or improperly 'capped' telomeres are major sources of genomic instability and have been linked to premature aging, hematological malignancies, and solid tumor formation. Thus, investigating how telomeres are maintained and how aberrant telomeres signal a DNA damage response, is essential to our understanding of cellular transformation. The objective of this proposal is to further define the mechanisms regulating the DNA damage response at telomeres and define how defects in this process promote genomic instability and ultimately, cancer progression. The studies described here will undoubtedly further our knowledge of the mechanisms involved in cancer progression and will lay the foundation for advances in disease diagnosis and/or treatment. The specific aims are outlined below. Specific Aim 1 will use a combination of biochemistry and cell biology to understand how the human POT1 protein protects telomere through two distinct functions. In mice, POT1 diverged into two genes encoding mPOT1a and mPOT1b proteins each possessing a unique function in telomere end protection. In this aim, I will use mouse mPOT1a and mPOT1b to determine how these two proteins differ in their ability to specifically repress ATR and how the single POT1 protein in humans accomplishes this function. Teasing out the functional domains in human POT1 will allow us to better understand how POT1 functions at telomeres and how mutations in POT1 may impair telomere end protection and contribute to tumorigenesis. Specific Aim 2 will use a combination of biochemistry and cell biology to determine how TRF2 represses ATM at telomeres. The goal of this aim is to understand mechanistically how TRF2 functions to inhibit ATM activation and ultimately, preserve genomic stability. Using new in vivo and in vitro assays, I will test the hypothesis TRF2 inhibits ATM activation by antagonizing binding of the DNA damage sensors at telomeric DNA. TRF2 is a key factor in telomere length maintenance and signaling, thus, dissecting the role of TRF2 in ATM inhibition will not only advance our current knowledge of how normal telomeres are maintained, but also how dysfunctional telomeres evoke a DNA damage response. The studies I have proposed here may shed light on how the telomere checkpoint is evoked and subsequently bypassed in cancers. Specific Aim 3 will use cell biology to investigate the role of the non-coding RNA TERRA in regulation of the DNA damage response at telomeres. TERRA is critical for maintaining genomic stability and is downregulated in a subset of human cancers suggesting that defects in TERRA contribute to telomere dysfunction and eventually, cellular transformation. The goal of this aim is to identify factors responsible for regulating the transcription, degradation, and/or localization of TERRA and to dissect the function of TERRA in maintaining genome stability. Despite my recent training in biochemistry, I will need 1-2 additional years of training to establish myself specifically as a telomere biochemist. This is a niche that is underrepresented in the field of telomere biology, and with additional training I feel I can make substantial contributions to the field. As an independent investigator, I will adapt my research from global DNA damage and genome maintenance, to telomere homeostasis and genome maintenance. In addition, I will continue to pursue research in the field of cancer biology and will begin to address the questions outlined in this proposal. Further defining the mechanism(s) regulating telomere stability will inevitably lead to a better understanding of cellular transformation and ma ultimately provide much needed therapeutic insight. I am eager to dissect the mechanisms regulating telomere homeostasis and would greatly appreciate the opportunity to conduct this research with the support of a K99 award. Receipt of this award would not only allow me to expand my research plan, but also establish myself as a primary investigator in the field of cancer biology. PUBLIC HEALTH RELEVANCE: Telomeres protect the natural ends of linear chromosomes from being recognized as sites of DNA damage. Given that DNA damage is a major source of genomic instability, the objective of this proposal is to further dissect the mechanisms regulating telomere maintenance and how defects in this process promote the genomic instability associated with cancer development.